Month: October 2014

In the movies, spooks and phantoms are often undead humans with unfinished business. But would you be afraid of a ghostly fruit fly?

In 1995, fruit fly researchers Christiane Nüsslein-Volhard and Eric Wieschaus were awarded a Nobel Prize for their research on development. They were interested in understanding how a fertilized egg develops into a complex organism, and were the first to show that development was controlled by genes. In their famous paper published in 1980, they found a small number of genes that were important in determining the body plan and formation of body segments in fruit fly larva.

Four years later, the same researchers published a set of papers on a group of genes that caused developmental defects in fruit fly embryos. When a gene in this group was mutated, the embryos died before the exoskeleton was created. The mutations somehow disrupted the formation of the embryonic cuticle, the protective outer layer that should form around the embryo. The researchers (not without a sense of humor after long grueling hours in the lab) dubbed them the Halloween genes. The genes earned their name not just because they mutated and killed, but because the mutant embryos took on a ghostly appearance. This resulted in gene names such as disembodied, spook, spookier, shadow, shade, shroud, and phantom.

Images of the cuticles of a normal fruit fly embryo and two with mutations in a Halloween gene. Modified from Gilbert, 2004

So what do the Halloween genes do? Since the 1980s, researchers have discovered that all of the Halloween genes are cytochrome P450 (CYP) enzymes involved in synthesizing a steroid hormone called 20-hydroxyecdysone (20E) from cholesterol. 20E is required for metamorphosis and moulting in arthropods such as insects and crabs. As a result, disruption of 20E synthesis in fruit flies blocked formation of the exoskeleton in embryos. Hmm… I had to look all that up.

CYP enzymes are found in most species and are involved in a very large variety of processes. In humans, they are involved in regulating hormones (among other things), including steroid hormones (just like in flies!). Steroid hormones are basically a group of steroids that act as hormones in the body, and they are synthesized from cholesterol (just like in flies!). Interestingly, testosterone and anabolic steroids, such as the ones that athletes may take, are actually steroid hormones. Thus, although mammals do not have 20E, they have other steroid hormones are important for development as well as reproduction, metabolism, and homeostasis, which allows cells to adapt to their changing environment. Because of these similarities, research in the Halloween genes may help us better understand how steroid hormones are synthesized in mammals.

The body’s biological clock is responsible for keeping track of time and synchronizing behavior with the environment, so that you feel alert during daylight hours and sleepy at night. This biological clock (also called the circadian clock or circadian rhythms) consists of three major parts:

The central pacemaker, which oscillates with a period of about 24 hours to keep time

Inputs to the clock, such as light or temperature, that synchronize it with the environment

Outputs from the clock, which signal to your body to indicate when it’s time to sleep, eat, etc

How does our body know when it’s time to feel sleepy or alert? The biological clock must have an output pathway for controlling sleep, but the mechanism of exactly how that happens is poorly understood. After analyzing the sleep patterns of thousands of fruit fly colonies with different genetic mutations, the authors of this paper found a group of flies that had trouble falling asleep at night. They called the mutated gene Wide Awake (wake for short) and discovered that while the gene wasn’t necessary for accurate functioning of the circadian clock itself, it was important for signaling a transition from wake to sleep at the end of the day.

During the day, GABA signaling from sleep circuits in the fly brain has little effect on LNvs (the wake-promoting clock cells). At dusk, increased amounts of WAKE proteins in clock cells leads to an increase in the number of GABA receptors on the membrane, thus increasing the cells’ sensitivity to GABA. This dampens LNvs activity, allowing flies to fall asleep at night.

So how does this gene translate clock information into a feeling of sleepiness? The authors looked to “clock cells” to answer this question. Clock cells are a group of cells that make up the central pacemaker. In mammals, this pacemaker is located in the suprachiasmatic nucleus (SCN). In flies, the central pacemaker includes a small group of neurons called LNvs, which promote wakefulness. During the day, activity in the LNvs causes flies to be awake and active. But at dusk, the amount of WAKE protein from the wake gene in these cells increases. WAKE makes the cells more susceptible to inhibition from a chemical messenger called GABA, which dampens activity. As the wake-inducing LNvs become more sensitive to GABA, they become less active, which allows the sleep circuits in the brain to exert more control over behavior so the flies can fall asleep. On the other hands, flies with a mutation in the wake gene were not getting enough GABA signal to quiet the LNvs at night, keeping them agitated and awake.

The authors identified a gene that seems to be a messenger from the circadian clock to the brain, telling it that it’s time to shut down and go to sleep at night. But how can these fruit fly findings help us humans? They also found the same gene in the SCN in mammals, suggesting that it plays the same role in other species as well. The finding that wake mutants had difficulty falling asleep after lights-off is reminiscent of sleep-onset insomnia in humans. Currently, the most common treatment for insomnia are drugs the mimic the effects of GABA in the brain. However, these findings suggest that the effectiveness of this treatment is limited if wake-promoting neurons are not sensitive enough to GABA signaling. Thus, future research in this area may focus on manipulating the wake gene and its proteins to develop a better treatment for sleep disorders such as insomnia in humans.

Over the past century, technological innovations have changed human society dramatically, undeniably for the better. But the advent of jet travel, round-the-clock manufacturing, and internet communication has also had a disruptive effect on our bodies’ circadian rhythms.

The word “circadian” comes from the latin words circa and diem, meaning “about a day”. Circadian rhythms—which are also referred to as our biological or internal clock—keep time at about 24 hours, and are found in most organisms including cyanobacteria, fruit flies, mice, and humans. In the wild, these biological pacemakers allow organisms to predict rhythmic changes in their environment, such as the day-night cycle of light and temperature variations. The clock helps animals to know when to sleep, eat, and when predators might be prowling.

Although the internal clock can remain rhythmic on its own, it regularly synchronizes with environmental changes such as light and temperature. This is important as light and temperature rhythms gradually fluctuate by the season, but sudden unexpected environmental cues can wreak havoc on our bodies until the clock can adapt and reset itself. The role of circadian rhythms in our lives can be best understood by seeing what happens when our clocks become desynchronized from the environment.

“Jet lag” is the first thing people usually think of for disruptions in circadian rhythms. When we travel through time zones, our body “remembers” the previous time zone, including the sleep-wake pattern, mental alertness and eating habits associated with it. For example, if you take a red-eye flight from California to New York, when you arrive your internal clock might tell you that it’s 3am, but outside, it’s time for breakfast. It can take days to adjust to the sudden shift in environmental cues in the new time zone, and during that time you feel tired and may find it “hard to think”.

In our ever increasing 24-hour society, disruption of normal circadian rhythms is commonplace. It can be caused by working long hours or with international partners, irregular shift-work, or even “social jet lag”, which is caused by having a different sleep schedule on the weekends than during the week. In addition, recent research suggests that teenagers experience a natural forward shift in their biological clock, causing them to become more alert later at night. During this period of life, getting up early every morning for school can be very disruptive.

When the circadian clock desynchronizes from the environment, it can have negative consequences to cognitive function and health. In the short term, a person can experience difficulties with learning and memory, mood disorders, immune system dysfunction, and generally reduced quality of life and well-being. Chronic disruption can lead to health problems such as obesity and diabetes, increased risk of cancer, heart disease, depression, and other conditions. To make matters worse, there are numerous sleep disorders that lead to loss of restful sleep. Could some of these be the result of dysfunction in the biological clock?

Circadian rhythm research is important for understanding the consequences of clock disruption and how it can be treated. There are several questions that need to be answered: How does the biological clock keep time? How does it synchronize with the environment and adjust to new time zones? Why do some individuals seem to have abnormal sleep-wake cycles, and how can these disorders be fixed?

How have fruit flies helped us understand circadian rhythms to answer these questions?

Although the existence of a circadian clock had been agreed upon for decades, until the 1970s no one knew anything about how it worked. Circadian rhythms are a complex behavior, and how do you even begin to piece together such a difficult puzzle? In 1971, a group of fruit fly researchers led by Dr. Seymour Benzer published a paper describing a gene named Period. They found three different mutations in the Period gene that caused flies to have faster, slower, or a complete absence of circadian rhythms. While it is now an accepted fact that all behaviors have a genetic component, at the time many were skeptical that genes could be involved in complex behaviors. Certainly they couldn’t believe that mutating a single gene would have an effect on something as complex as circadian rhythms.

Now more than 40 years later, research in circadian rhythms has made it the best understood complex behavior at the genetic and molecular level. Many of the pioneering experiments on circadian rhythms were performed in fruit flies because they are such an easy-to-use genetic animal model, and the genes and processes involved in mammalian circadian rhythms were often first identified in fruit flies. Once researchers understood the basic components of the clock in flies, they were able to apply that knowledge to the more complex mammalian systems in later studies. The core mechanism for the biological clock is shared between flies and mammals, which spans over 600 million years of evolutionary time!

How does the biological clock keep time?

Click on the picture for a bigger version. Circadian rhythms in both flies and mice are controlled by two negative transcriptional feedback loops. Although there are various differences between the species, the structure is similar. Modified from Hardin, 2000

Since the 1970s, dozens more clock genes have been identified including Timeless, Clock, and Cycle. Researchers have learned that the clock keeps time via daily fluctuations of clock-related proteins which interact in what is called a “negative transcriptional feedback loop”. There are two such loops: the Period/Timeless (Per/Tim) loop and the Clock/Cycle (Clk/Cyc) loop. In the Clk/Cyc loop, levels of both Clock protein and Cycle protein are high during the day, and then decrease after dark. When there is enough of each protein in the cell, Clock and Cycle proteins bind together and attach to strands of DNA to activate the Period and Timeless genes. This allows the cell to create Period and Timeless proteins from the DNA’s instructions. Thus, over the course of the day, the cell starts to make plenty of Period and Timeless proteins, but light-activated mechanisms (involving a gene named Doubletime) in the cell break them down. When night falls, however, levels of these proteins can begin to accumulate. The Per/Tim loop is therefore the reverse of the Clk/Cyc loop in that there are low levels of proteins during the day which increase after dark. When the levels of each protein are high enough, Period and Timeless bind together and inhibit Clock and Cycle, effectively deactivating the Period and Timeless genes so that no new protein is being made. As the next day begins and light begins breaking down the Period and Timeless proteins again, the cycle restarts. This is a simplified explanation of the system, and there are many more genes involved (especially in the mammalian version), but this two-loop clock is found in flies, mammals, and other species, suggesting that it is the optimal system for timekeeping.

For a great visualization of how the molecular clock works, check out the graphic on this website under “How do clock genes work”. This page also gives more information about the mammalian circadian clock if you’re interested.

More recent research has also begun to focus on how networks of cells work together to keep each other synchronized. Although each cell has its own running internal clock, it can become desynchronized by error or damage. Researchers have found that clock cells communicate with each other to maintain an overall synchronized circadian rhythm.

How does the clock synchronize with the environment and adjust to new time zones?

Although circadian rhythms continue even in the absence of environmental stimuli (such as in complete darkness and stable temperature), the clock can also synchronize with the environment based on cues such as light, temperature, or other rhythmic stimuli. The actual circadian period is slightly longer than 24 hours, but the environmental length of day can vary based on location and/or season. Researchers have found that the clock can be reset by unexpected light pulses or fluctuations in temperature, but how do external cues reset the clock? While not much is known yet about the effects of most cues, scientists have made extensive progress in understanding how the clock synchronizes to light.

In fruit flies, light entering the brain through the fly head and eyes can break down the Timeless protein, so levels of this protein can’t start to increase until after dark. This contributes to the normal cycling behavior. But when an unexpected light pulse occurs, such as too early in the morning or too late at night, degradation of Timeless causes the brain to think that the next “day” cycle has begun and resets the circadian clock. There are other genes involved in this process as well, such as Crytochrome and the aptly-named Jetlag. In 2006, it was found that a mutation in the Jetlag gene causes defects in flies’ ability to adapt to a new light-dark cycle. A shared gene in humans might be responsible for differences in individuals’ ability to adapt after flying to a new time zone.

The future of circadian research: Why do some individuals have abnormal sleep-wake cycles, and how can circadian disorders be fixed?

By increasing our understanding of how the circadian clock ticks away the time and responds to environmental cues, we may be able to design drugs to treat jet lag, reset circadian rhythms, or even reverse the consequences of a desynchronized clock.

Research may even help scientists better understand human sleep disorders such as narcolepsy or insomnia and identify underlying circadian components. For example, humans with a sleep disorder called Familial advanced sleep phase syndrome (FASPS) have been described as having a shifted circadian rhythm. Though they have a normal sleep structure, everything is pushed forward by about 4-6 hours. As a result, individuals tend to wake up well before sunrise (around 1-3am) and feel compelled to sleep around 6-8pm. A similar shifted sleep schedule has been reported in Doubletime fruit fly mutants and tau mutants in hamsters. Eventually, researchers found that FASPS is caused by mutations in similar clock genes in humans called PER2 and CSNK1D. A better understanding of the role these genes play in circadian rhythms could help scientists develop medicines to treat this disorder.

When it comes to genetic research, fruit flies take the spotlight. They are often used to study specific genes, and researchers who find new genes get the privilege of naming them. The best way to study a gene is to mutate it and see what happens when the gene’s function is lost. As a result, genes are usually named after the phenotype caused by the mutation. For example, one of the genes I work with is amnesiac. As you can probably guess, flies with a mutation in the amnesiac gene can’t form long-term memories!

This naming convention can often be very confusing for first-time fly students. One of the first genes we learn about is the white gene, which codes for eye color. Flies with a normal version of the white gene (usually written as w+ ) actually have red eyes, while flies with a mutation in the white gene ( w– ) have white eyes.

Over the years, fly scientists have amused themselves by choosing hilariously descriptive names for newly discovered genes. Here are some of my favorites (these are real scientific gene names):

Cheapdate– mutants have decreased resistance to the effects of alcohol

Hangover– mutants can’t develop a tolerance for alcohol

Dunce– mutants have learning deficits

Tinman– This gene regulates the development of a heart, so flies with a mutation in this gene can’t grow a heart. Named after the character in Wizard of Oz

Ken and Barbie – mutants do not develop external genitals

Swiss cheese – mutants have holes in their brain

I’m not dead yet – mutants live twice as long as normal flies, a reference to Monty Python and the Holy Grail

Groucho Marx – mutants develop an excess of facial bristles

Out Cold – mutants lose coordination at cold temperatures

Clumsy – mutants have trouble walking

Fly scientists have been getting away with choosing funny (and sometimes politically incorrect) gene names for decades. But over 75% of fruit fly genes are shared with humans, so not only would some of them eventually be found in humans, but they’d also be associated with a genetic disorder. And that’s where fly scientists can get themselves into trouble. For example, several years ago a gene named Hedgehog made headlines when it was found in humans.

The Hedgehog gene was discovered in 1980, and was given its name based on the observation that a mutation in this gene led to spiky-looking larvae. Three versions of this gene were later found in humans, one of which was named Sonic Hedgehog (SHH) after Sega’s video game character. Unfortunately, this gene has since been linked to a human developmental disorder called Holoprosencephaly, in which an SHH mutation leads to severe brain, skull, and facial defects. Doctors soon began to protest having to explain the joke name to patients with extreme medical conditions.

In 2006, the Human Genome Organization Gene Nomenclature Committee stepped in and changed some of the most offensively named genes. Another gene that was targeted was Lunatic Fringe, which was originally given its name because mutant flies had defects on the edges of their wings. In humans, however, mutations in the gene (which was renamed LFNG) leads a hereditary spinal defect called Jarcho-Levin Syndrome.

But despite these naming blunders, fruit fly research on these genes has and will continue to help in the search for treatments for these genetic disorders. And fly scientists haven’t stopped taking the opportunity to come up with fun names for newly discovered genes. Earlier this year, the Dickson fly lab published a report on “Moonwalker” flies: flies that couldn’t change direction and were permanently stuck walking backwards.

Video: The left side shows a fly walking normally. The right side shows a “moonwalking” fly unable to walk forwards.

How can we slow or even halt the steady march of aging? In a previous post, I reviewed a paper that asked “What causes aging?” (the prevailing hypothesis is that aging is caused by accumulating cell damage). Understanding why we age is important for developing ways to interfere with the process. But there are other ways to study aging in the hopes of one day developing the mythical “elixir of life”. A recent fruit fly paper published in Cell Reports by the Walker lab instead asked, “How does caloric restriction increase lifespan?”

Scientists discovered a long time ago that caloric restriction can extend lifespan in a wide range of animal models, including rats and fruit flies. But the diet is severe and leads to side effects including reduced energy levels and sensitivity to cold. Who wants to live longer in such misery? Decreased food intake itself can’t be the cause of increased lifespan (it does seem rather counterintuitive), so what is going on?

The authors found that activating a molecule called AMPK could increase lifespan just like dietary restriction, without all those miserable side effects. Normally, AMPK is activated when a cell needs more energy (such as when the animal isn’t eating enough calories). AMPK then triggers a process called macroautophagy, which means that components and molecules inside of cells are broken down and recycled for energy. This usually happens when the cell needs more energy than it is being provided, and the first things to go are old, unnecessary, and damaged cellular components. By artificially activating AMPK, the authors tricked the cells into breaking down more of this “cellular garbage”, slowing the accumulation of damage that leads to aging.

Perhaps the most exciting finding was that the authors could extend the lifespan of adult flies just by activating AMPK in the intestines. This manipulation slowed aging not just in intestinal cells, but also in brain tissue and even muscles. In fact, older flies even showed improved climbing ability. Although there is much work left to be done, these findings give hope that one day, aging can be slowed in adult humans just by swallowing a pill that activates AMPK in our gut.